Metabolic flux analyses (MFA) are becoming increasingly important as a central element of modern systems biology. Using MFA, the rates of metabolite movement within complex intracellular networks are quantified. In interaction with proteome, transcriptome and genome analyses, MFAs allow genetic and environmental effects on the growth of plants and other organisms to be demonstrated.
In the quantification of metabolic pathways via MFA, the labeling of compounds which are taken up by the plant has proved expedient (Szyperski (1998) 13C-NMR, MS and metabolic flux balancing in biotechnology research. Quart. Riev. Biophys. 31:41-106). Substances which are suitable in this context are radioisotopes such as, for example, 14C, 3H or 32P, or stable isotopes such as, for example 2H, 13C, 18O, 15N. The labeled compounds which are taken up by the plant, for example labeled carbon dioxide, follow intracellular metabolic pathways, whereby the labeled isotopes are distributed and incorporated into the intracellular metabolites or metabolic end products of interest (for example protein, starch, lipid, cell wall). The labeling in metabolites or metabolic end products can subsequently be detected via mass spectrometry or nuclear magnetic resonance. The typical labeling patterns of intermediates of the central metabolism or its end products, therefore, generate an “isotope fingerprint”, by means of which the actual flux distribution may be calculated.
The experimental determination of systemic metabolic fluxes using 13C isotopes has long been used for studying a variety of organisms such as, for example, Penicillium chrysogenum (Christensen and Nielsen, 2000), Escherichia coli (Fischer and Sauer, 2003; Zhao et al., 2004), various yeasts (Blank et al., 2005), Bacillus subtilis (Sauer et al., 1997), Corynebacterium glutamicum (Marx et al., 1996), Synechocystis (Yang et al., 2002), Methylobacterium extorquens (Van Dien et al., 2003), soybean embryos (Sriram et al., 2004) and oilseed rape embryos (Schwender et al., 2004).
In contrast to traditional 13C labeling methods, the isotopic non-stationary metabolic flux analysis (“INST 13C-MFA”), also referred to as dynamic metabolic flux analysis, generates time profiles of the labeling patterns. Here, the transient labeling information of the metabolites is used for determining the in-vivo fluxes (for an overview, see Nöh and Wiechert, 2011, Appl. Microbiol. Biotechnol., 91, pages 1247-1265), which is not possible when using stationary methods, in which only the post-saturation 13C accumulation is measured. The dynamic method permits the metabolic flux analysis of photoautotrophic systems under physiological conditions with the use of CO2. This analysis is not possible with stationary methods, for which reason in-vitro organ cultures are generally used for metabolic flux analyses of plants. In-vitro organ cultures are highly artificial systems, therefore the analysis of an intact plant under physiological conditions is advantageous.
Processes and apparatuses which are optionally suitable for isotope labeling are known from the prior art. For example, DE1949001 describes an apparatus for adjusting the atmospheric humidity in a plant growth chamber. A shortcoming of this type of arrangement is the necessity of an artificial light source since nontransparent material is used for the walls of the housing. Furthermore, the apparatus disclosed in DE1949001 lacks a material for absorbing carbon dioxide before the labeling is taken up, and also suitable devices for supplying CO2.
An apparatus for regulating and determining the carbon dioxide content of a growth chamber by means of a regulated supply and removal of carbon dioxide and an apparatus for absorbing carbon dioxide are described in DE 1773320; however, the chamber described in DE 1773320 takes the form of a system for recording the total carbon dioxide turnover of plants (or other organisms such as lichen)—an intervention in the sense of continuous sampling, as required for the analysis of metabolic fluxes and made possible by the labeling chamber which is provided in accordance with the invention with a lock is not provided.
The technological background of the present invention is furthermore described in U.S. Pat. No. 3,673,733, where, likewise, the previous removal of nonlabeled carbon dioxide by means of the apparatus described in U.S. Pat. No. 3,673,733 is not possible.
U.S. Pat. No. 5,341,595 discloses a chamber for analyzing the growth of plants, where, likewise, the previous removal of nonlabeled carbon dioxide by means of the apparatus described in U.S. Pat. No. 5,341,595 is not possible. Moreover, the apparatus disclosed in U.S. Pat. No. 5,341,595 lacks a system for uniform aeration.
Chen et al. (Proteome Science 2011, 9:9 http://www.proteomesci.com/content/9/1/9) discloses a sealed plant growth chamber by means of which humidity, pressure, temperature and 13CO2 concentration can be controlled and kept constant. In contrast to the present invention, the chamber described by Chen et al., however, does not include a lock which, in a dynamic MFA, would allow the continuous sampling without a gas exchange between the inside of the chamber and the external environment taking place.
This is the reason why, in Chen et al., the plants are maintained for several weeks in a pure 13CO2 atmosphere, whereby they are almost completely 13CO2-labeled. Thereafter, the chamber is opened and aired in a surge-like manner, whereupon samples are taken promptly. In other words, what is measured is not, as is possible in the chamber according to the invention, an accumulation of 13C in the plants/metabolites, but a depletion of the 13C, or an accumulation of the 12C from the surrounding atmosphere. Therefore, the method described by Chen et al. is considerably more costly since an inordinately higher amount of 13CO2 is required.
Moreover, the process described in Chen et al. is a much greater deviation from natural/physiological conditions than the exposure of the plants to be labeled with 13CO2 in the process according to the invention, which exposure is only short since the plants remain for a prolonged period in a sealed system in which an artificial atmosphere prevails. It is known that the photosynthetic utilization, by plants, of 12CO2 and 13CO2 differs.
Plant growth chambers which are suitable in principle for the 13CO2 labeling of plants are supplied commercially under the product name “BioBox” by GMS Gaswechsel-Messsysteme GmbH Berlin; here, again, artificial illumination is employed, and only the costly variant of labeling up to the 13CO2 saturation followed by measuring the 13CO2 decrease in the various metabolites is possible. Here, too, the plants to be labeled have to remain in an artificial atmosphere and under artificial illumination conditions over a substantial period.
The direct measurement of the incorporation of 13C as per the INST-C-MFA method is known from the prior art for single-celled organisms or liquid cultures of plant cells. Thus, for example, Young et al. (2011, Metabolic Engineering 13, pages 656-665) show a carbon flux chart of the single-celled cyanobacterium Synechocystis where the intracellular 13C distributions were used for calculating metabolic fluxes under photoautotrophic conditions. The high sampling frequency required herefor may be attained for example by withdrawing a sample volume by means of a stopcock, if using liquid cultures.
An apparatus for rapid sampling and therefore measuring of the 13CO2 incorporation of higher plants, which apparatus would make possible a considerably more efficient, more precise and more natural experimental set-up, is not known from the prior art.
Accordingly, it has so far not been possible to carry out such measurements in particular on intact crop plants such as maize, rice, soybeans or oilseed rape.
An object of the present invention was therefore to provide a labeling chamber for plants, by means of which labeling chamber reliable dynamic metabolic flux analyses may be carried out in plants without an undesired gas exchange between the inside of the chamber and the environment affecting the results measured. The prior-art systems do not allow any continuous sampling without adversely affecting the climate of the chamber in the long term. In addition, the prolonged 13CO2 saturation achieved in the known systems does not correspond to the natural state. Moreover, less 13CO2 is consumed (thus resulting in lower costs) in the apparatus according to the invention than in the inverse method described by Chen et al.
This object was achieved by the subject matter of the present invention, which relates to an isotope labeling chamber for labeling metabolites in an organism, preferably in a plant, comprising a reactor chamber (1) and an air regulation chamber (2), wherein the reactor chamber (1) comprises the following components:
optionally, a housing frame (3), housing walls (4), at least one injection valve (5), where at least one housing wall (4) can be opened fully and/or in part and where at least one housing wall has a lock (6),
and furthermore wherein the air regulation chamber (2) comprises the following components:
a temperature-regulating unit (7), an air humidification unit (8) and a gas absorption unit (9).
The temperature-regulating unit, the air humidification unit and the gas absorption unit are connected to the upper reactor chamber in each case via passages and/or tubes and, therefore, together with the reactor chamber constitute circulations and air/gas exchanges which are independent of each other.
The reactor chamber (upper compartment: housing walls and upper part of the frame) is made as a module, in other words may be varied, only the air regulation chamber (lower compartment) being permanently provided with technology so that reactor or plant chambers of different sizes may be combined with the air regulation chamber.